Mounting data suggests that both neutralizing and non-neutralizing binding antibodies are associated with protection against HIV infection. Induction of these antibodies therefore is now a high priority for HIV-1 vaccine development, and nonhuman primate (NHP) challenge models play a critical role in the pre-clinical evaluation of the effectiveness and breadth of vaccine-induced responses. At least nine different genetic subtypes and a growing number of circulating recombinant forms of group M HIV-1 account for the majority of new infections worldwide, with subtype C viruses predominating in countries that carry the heaviest burden of infection. Reliable NHP challenge stocks representing globally circulating HIV-1 strains therefore are needed to determine if vaccine-induced immunity has activities against multiple variants within a single genetic subtype as well as across subtypes. Understanding the basis for subtype differences in pathogenesis and transmission will also be important in the design of effective prevention strategies. In the current funding cycle, we fully characterized the pathogenic subtype B R5 SHIVSF162P3N infection model for vaccine studies, and successfully constructed molecular clones of this isolate that induce acute CD4+ T cell depletion in the gut, progression to AIDS with coreceptor switch coincident with peripheral CD4 decline and V3 loop sequence change. More importantly, we generated a series of lineage-related, mucosally transmissible subtype C R5 SHIVs that are also capable of AIDS induction, with neuropathological development and coreceptor switching at terminal disease. The experiments proposed in this competitive renewal application aim to expand on these successes by constructing subtype C R5 SHIV molecular clones as well as additional non-subtype B SHIVs that recapitulate the immunopathogenesis of HIV-1 in humans as challenge viruses to improve the utility and predictive value of the SHIV/macaque models. We will also use the pathogenic subtype B and C R5 SHIVs generated as tools to address if there are subtype differences in HIV-1 replication, immunology and disease. Three hypotheses will be tested in experiments with the following specific aims: Aim 1: Construct subtype C R5 SHIV molecular clones with dynamics of infection and pathologies akin to the human infection. We hypothesize that these molecular clones will be important for studies aimed at dissecting the evolutionary changes in env that influence disease outcomes and vaccine escape. Aim 2: Generate and characterize additional pathogenic non-subtype B R5 SHIVs using sCD4-sensitive transmitted/founder Envs. We hypothesize that the use of sCD4 sensitive Envs that bind CD4 efficiently provides a better chance of success in generating highly infectious and pathogenic SHIVs. Aim 3: Compare immunopathogenesis of subtype B and C R5 SHIV infection in RMs. We posit that such studies may lead to the identification of biologic and/or immunologic factors that impact HIV-1 subtype spread and disease.